Disposable bioreactor system

ABSTRACT

A cylindrical or annular polymeric bioreactor is disclosed which provides enhanced mixing and aeration of the growth medium while simultaneously offering reduced mechanical shear force.

RELATED APPLICATIONS

This application claims priority from co-pending, commonly assignedprovisional application Ser. No. 60/927,274, filed 2 May, 2007.

FIELD OF THE INVENTION

This invention relates to an improved design for a disposable bioreactorvessel suitable for cell culture and fermentation.

BACKGROUND OF THE INVENTION

The increasing popularity of disposable or single use bioreactors forupstream processing has been recently noted in several studies, and canbe understood by considering a typical biotech manufacturing facility.The infrastructure required to implement a facility using traditionalglass/steel bioreactors is substantial, and the time and expenserequired to construct it can be immense. The requirement that both thebioreactor itself, and also the ingress and egress tubing, utilize inertmaterials such as 316L electro-polished stainless steel requires a largeinitial investment. Additionally, the size and form factor of disposablebioreactor vessels generally lend themselves to easier storage andre-configurability when compared with traditional, rigid glass/steelsolutions. Other advantages are the cost and time savings overtraditional designs, the reduction in preparation and sterilizationtime, the reduced need for purified water for cleaning the vessel aftera run, and the significantly reduced post run maintenance time.Additionally, single use bioreactors and the associated plastic tubinglend themselves to being re-configured and validated quickly andefficiently as manufacturing or process requirements change. Although anumber of different styles of single use bioreactors have been conceivedand introduced into the marketplace, two types currently predominate.

The first type of single-use bioreactor is generally referred to as the“pillow” or “rocker” bag style, and is described, for example, in U.S.Pat. No. 6,190,913. This style of bag has been constructed from avariety of different polymeric materials, but low density polyethyleneand ethylene/vinyl-acetate copolymers are currently the most popularmaterials for at least the innermost layer which contacts the aqueousgrowth medium. This type of disposable bioreactor utilizes a wave motioninduced by movement about a single axis to both mix and sparge (aerate)the contents of the bioreactor.

Another style of bioreactor bag is often referred to as a “liner style”and substantially mimics the function and form of a traditionalglass/steel bioreactor. A disposable polymeric bag is used as a linerinside a generally cylindrical glass/steel tank and generally uses animpeller to mix the contents of the bioreactor vessel. This type ofsystem has been commercialized by several manufacturers (see e.g.,Published US Patent Applications 2005/0272146 and 2005/0239199). Thedisposable liner type of bioreactor bag has proven popular for processdevelopment or pilot runs using growth medium volumes of 25 liters ormore.

Both styles of disposable bioreactors have undergone some scrutiny inorder to judge their efficacy in comparison to traditional glass/steelbioreactors. However, statistically rigorous analyses are apparently notavailable to date. Irrespective of the style of the disposablebioreactor, the inner surface of the polymeric bioreactor bag needs tobe both biologically inert and also not prone to leaching nutrients fromthe growth medium or from the polymer into the growth medium (seeKadarusman et. al, Growing Cholestero-Dependent NSO Myeloma Cell Line inthe Wave Bioreactor System: Overcoming Cholesterol-Polymer Interactionby using Pretreated Polymer or Inert Fluorinated Ethylene Propylene,Biotechology Progress, 2005, 21, p. 1341). Additionally, the liner needsto be chemically stable under the optical illumination often used tofacilitate cell growth.

Whether a bioreactor is of traditional design or a modern disposableformat, the basic function of a bioreactor is to provide a controlledenvironment in order to achieve optimal growth/product formation in thecell or microbe that is present in the aqueous bioreactor growth medium.The traditional glass and steel bioreactors used in batch processes wereproven to be effective in the course of antibiotic production in the1950's, and still remain the dominant paradigm in the fermentationindustry. Criteria for optimizing bioreactor design have been enumeratedbefore (see A. Margaritas and J. B. Wallace, Novel Bioreactor Systemsand Their Applications, Nature Bio/Technology, May 1984, p. 447.)According to Margaritas et al, some of the basic criteria required tocharacterize and understand bioreactor performance are:

-   1. Mass and heat transfer and hydrodynamic characteristics of the    bioreactor,-   2. Potential for bioreactor scale-up, and-   3. Capital and operating costs of the bioreactor

Given the extensive existing knowledge regarding the design andperformance of traditional glass/steel bioreactors, it was considereddesirable to mimic their design as much as possible in theimplementation of a disposable bioreactor system. Examination of currentproducts reveals that this approach has been utilized, but with onlymixed results. An example of this prior art style of product, as shownin FIGS. 1a and 1b , employs a liner made of a bio-compatible, flexiblepolymeric material set inside a vessel which provides structuralsupport. The end result is a disposable liner which assumes a physicalform very similar to that of a traditional (generally cylindrical)bioreactor. The use of an impeller and sparger completes the analogy toa traditional glass/steel vessel. Therefore, the performance of a linertype disposable bioreactor can be measured against the list of criteria1 through 3 above, and one can get a reasonable idea of the performancethrough comparison to the performance of similar size and shapetraditional glass/steel systems. Given the analogy to known bioreactorshapes and sizes, impeller design and placement, the mixing andoxygenation rates can be approximated. Additionally, traditional scalingarguments can be formulated and implemented, and known techniques ofcomputational fluid mechanics can be applied.

The “pillow” bag style of disposable bioreactor also utilizes bags madeof biocompatible films. An example of this design is shown in FIG. 2(see V. Singh, Disposable Bioreactor for cell culture using wave-inducedagitation, Cytotechnology 30, 1999, p. 149.). Most proponents of the“pillow” bag stipulate that traditional bioreactors and single-usebioreactors that emulate the traditional glass/steel bioreactor using adisposable liner limit the yield obtainable in mammalian cell growthruns due to impeller induced sheer stress of the cells during mixing andsparging. The “pillow” bag style of disposable bioreactor bag has noimpeller, and utilizes a rocking motion to mix and sparge themicro-organisms contained within the bag. It is claimed that thisrocking motion is gentle on the cells and does not cause sheer stresswhich can easily damage mammalian cells (G. Kretzmer and K. Schugerl,Journal of Applied Microbiology and Biotechnology, Vol. 34, No. 5,613-616 (1991)). The boundary conditions set forth by the bag, combinedwith the rocking motion are alleged to adequately mix and aerate theaqueous culture medium when mammalian cells are used. However, it is notentirely clear that this is the case or that this style of bioreactor isefficacious for non-mammalian cell processes, such as bacterial ormicrobial fermentations that require more oxygen and/or can have highlyviscous contents (reaction medium).

As shown in FIG. 2, the most common commercial versions of the pillowbag style single-use bioreactor utilizes one or more ports on the top ofthe bag to bring in the air or oxygen, and then utilizes another(output) port in relatively close proximity to the input port to ventthe gas(es) produced by the bioprocess. It is likely that in many casesthe incoming sparging gas is vented too quickly so that sub-optimaloxygenation occurs. For this type of bag, no evidence of anunderstanding of the performance criteria 1 through 3, as set forthabove, is found in the literature. Published data generally indicatesthat the total cell density achieved in this style of bioreactor isgenerally less than (and at best, equal to) that achieved in smallshaker flasks. It is an object of the present invention to provide adesign for a single-use bioreactor vessel which ensures adequate mixingof the ingredients in the growth medium, and sufficient aeration forcells or microbes which require significant oxygen, but at the same timedoes not cause destructive shear stress and is scalable to comparativelylarge volume runs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the prior art type of disposable bioreactor system (e.g.,Hyclone/Thermo-Fisher) that utilizes a disposable polymeric liner andprovides structural support for the liner.

FIG. 2 shows a prior art “pillow” disposable bioreactor bag of flexiblepolymeric material.

FIG. 3a shows a three-dimensional view of a bioreactor in accordancewith the present invention having vertical side walls and which istoroidal in shape.

FIG. 3b shows two-dimensional views of a toroidal bioreactor inaccordance with the present invention as depicted in FIG. 3a and showsalternative embodiments of the vessel having either a circular (12) orrectangular (11) cross-section.

FIG. 3c shows how the bioreactor of the present invention achieves itsenhanced mixing via orbital motion (in this case moving counterclockwise indicated as 16) which creates a travelling wave of the growthmedium present in the bioreactor vessel (17).

FIG. 4 shows a view of the walls of the preferred embodiment of thebioreactor vessel of FIGS. 3a and 3b where sparging is accomplishedthrough the walls of the vessel. The walls are shown as corrugated inorder to enhance mixing.

FIG. 5 shows a view of an alternative configuration for a bioreactorvessel in accordance with the present invention where the bioreactor istoroidal but oval in cross-section rather than circular. Again, thevessel can have a cross-section that is either substantially rectangular(5.2) or circular (5.3).

FIG. 6 shows an embodiment of the present invention which provides formulti-directional motion (i.e, both orbital and rocking) of thebioreactor vessel of the present invention shown in FIG. 3a , with the Xand Y axes shown.

DESCRIPTION OF THE INVENTION

The most desirable single-use bioreactor solution would be one thateffectively meets criteria 1 through 3, as set forth above, while at thesame time requiring the fewest moving parts. Minimizing the number ofmoving parts will also tend to yield the least expensive and mostreliable overall solution. A novel design for a polymeric bioreactorvessel in accordance with the present invention which uses a minimalnumber of parts is shown in FIGS. 3 through 6.

In FIG. 3a , a cylindrical bioreactor vessel 1 having a substantiallyrectangular cross-section is shown having a hollow core 6, so that thebioreactor is a toroid. Although the design shown in FIG. 3a is shown ashaving substantially vertical walls, this is not an essential aspect ofthe invention and a toroidal (donut) shaped vessel not having verticalsurfaces is also suitable, as shown in FIG. 3b in cross section as 12.Alternatively, only one wall (either inner or outer) may be vertical andthe other curved.

The vessel can suitably be of a rigid biocompatible polymer.Alternatively, the vessel can be fabricated in whole or in part of aflexible (non-rigid) polymer since the hydrostatic pressure of theliquid reaction (growth) medium present within the vessel together withthe pressure of the sparging oxygen or oxygen containing gas will enableeven a bag made of non-rigid polymer to substantially retain its annularshape. Suitable rigid polymers include, but are not limited to; USPClass VI approved polycarbonate and polystyrene. Suitable flexiblepolymers include, but are not limited to, low density polyethylene andethylene/vinyl acetate copolymer.

In FIG. 3a , element 2 is a platform equipped with an electric motor orother drive means (not shown) which moves in an orbital motion asfurther shown in FIG. 3c . This orbital motion, combined with thetoroidal shape of the bioreactor, sets up a traveling wave in thebioreactor growth medium. This traveling wave provides both effectivemixing and oxygenation of the contents. It should be noted that in thecase of a torioidal vessel whether having vertical or curved walls, theliquid motion will follow a pure traveling wave. In both cases, theexact liquid motion can be modeled using computational fluid dynamics.

Additionally, single-use bioreactor vessel 1 can suitably be equippedwith one or a plurality of input ports (one shown as 4 in FIG. 3a ) forprocess feedstock inputs (e.g.: pH buffers, glucose etc.), and/or alsofor pre-inserted and pre-calibrated sensors (e.g., to measure dissolvedoxygen, pH, dissolved CO₂ and the like). These sensors can be eithertraditional electrochemical sensors and/or disposable and pre-calibratedoptical sensors. It is generally more efficacious to have the sensorspre-calibrated in order to avoid breaking the sterile barrier on thebioreactor. Optical probes are available that can be both pre-calibratedand can be gamma irradiated while mounted in the bioreactor. The abilityto use gamma radiation allows the pre-calibrated sensors to be insertedin the bioreactor and the entire assembly subsequently sterilized. Port5 denotes the exit port for removal of gaseous reaction by-products. InFIG. 3a 7 denotes a light source such as a bank of visible or UV lightemitting LEDs that can be utilized to promote the growth of plant oralgae culture or transiently activate certain genes for multi-drugproduction from a single cell platform The lighting is shown on the top,but this is not a necessity; the illumination can additionally oralternatively come from the sides and/or even the bottom of the vesselprovided only that the growth media receives enough illumination toenhance photosynthesis or gene activation depending on the purpose ofthe growth run and the cell being grown. Ideally the LEDs will have aspectrum that is matched to the absorption spectrum of the species beinggrown. LEDs are now available from the ultra-violet through the nearinfrared so matching to the absorption spectrum of the species understudy can be easily done. Additionally, the surface of the bioreactor onwhich the LED's are mounted needs to be sufficiently transmitting inthis region to allow the light to pass through to the interior of thebioreactor

The bioreactor vessel walls (outer and optional inner surfaces) define astructure that will have a resonant frequency which is determined by theparticular configuration (size and shape) of the bioreactor. Theresonant frequency of the bioreactor can be readily calculated knowingthat the traveling wave must reproduce itself in phase every round trip(Hydrodynamics, Horace Lamb and Russ Caflisch, First CambridgeUniversity Press, 1997). Once this frequency has been determined thebioreactor can be rotated with this circular frequency to thereby set-upresonant traveling wave motion of the fluid (growth medium) inside. Thewave amplitude will be chosen depending on the level of mixing andagitation needed.

In FIG. 3b the top view, 10, of the bioreactor is shown along with thesquare or alternatively circular cross sections 11 and 12 respectively.

It is possible to further enhance the mixing and sparging efficiency ofthe bioreactor vessel of the present invention shown in FIG. 4 byproviding a textured surface on the inner floor (bottom) and/or wallsurfaces of the vessel (i.e., a surface which is in contact with thegrowth medium). This texturing can be achieved using baffles, ridges,bumps and/or other upstanding protuberances as shown in FIG. 4, whichare preferably pre-molded on all or a part of the inner surface of thevessel. The sparging of the reaction medium is preferably accomplishedin this design by bringing the oxygen or air into the bioreactor througha multitude of orifices in the floor and/or walls of the vessel. Theseorifices can be in the protuberances in the texturing or separate fromthem. In FIG. 4, the flow direction of the fluid in the annular vesselis indicated as 4.1, (which corresponds to arrow 3 in FIG. 4), thetextured surface on the inner wall and/or floor surface of the vessel(the textured surface being shown here for simplicity to have anapproximately sinusoidal shape pattern) is indicated as 4.2, and thesparge orifices are indicated as 4.3.

The optimal patterning (e.g., size, shape and frequency) will be afunction of the size of the reactor, the velocity, viscosity, and natureof cell platform and its associated optimized growth medium. Theparticular patterning which provides optimal agitation can be determinedthrough finite element analysis studies or through empirical experiment.These studies generally include mixing studies as a function of time ornumber of agitation cycles. Additional computational studies that employHenry's law (p. 384, General Chemistry, 2^(nd) Edition, Donald A.McQuarrie and Peter A. Rock, W. H. Freeman and Company, New York, 1987)to model oxygen transfer or calculate the oxygen transfer rate arepossible. These studies require the finite element analysis code to takeinto account the surface area of the bubbles created during sparging.For example, a higher number of bubbles having decreased size willincrease the surface area available for oxygen transfer.

In FIG. 4, the sparging gas (normally air or oxygen) is suitably broughtto the bioreactor using tubing 4.3 which preferably will have a fluiddiode or hydrophobic filter at or near its end that allows the sparginggas to flow into the bioreactor, but prevents the liquid (reactionmedium) from draining out of the bioreactor vessel through said tubing.The aforementioned texturing of the floor and/or walls therefore doesnot preclude bringing the sparge gas into the vessel through the floor,top, and/or walls of the vessel. This type of sparging, combined withthe traveling wave motion induced by the orbital movement of the vessel,ensures that the cells or microbes in the bioreactor have access tosufficient feed and oxygen to achieve optimal total cell density and/ormaximize product yield.

It should also be noted that the motion in direction FIG. 3a is onlyshown as orbital, but can also involve rocking in either the X or Y axis(or both) and thereby be a two-dimensional or three-dimensional motion,which can be readily tailored to work in conjunction with the bioreactordimensions. Such multi-directional motion is shown in FIG. 6 where 6.1and 6.2 are the plane of the platform when rocked about axis 6.3. Theplatform holding the bioreactor can also be orbited in one directionaccording to 6.4, or agitated in both directions as per 6.5. This motioncan also be a reciprocating motion where the direction of rotation ofthe motor which causes the vessel to orbit is periodically reversed.This periodic reversal in the direction of the vessel's orbit can beused to cause more vigorous agitation if/when required during the growthprocess.

We claim:
 1. An apparatus comprising: (a) a hollow toroidal one-piecepolymeric bioreactor vessel comprising at least one port configured toreceive at least one pre-inserted and pre-calibrated sensor to monitor abioprocess carried out in said hollow toroidal one-piece polymericbioreactor vessel; and (b) a drive configured to move the hollowtoroidal one-piece polymeric bioreactor vessel such that there isbuild-up of a resonant frequency traveling wave of fluid orbiting in onedirection in an interior of the hollow toroidal one-piece polymericvessel when a particular amplitude and orbital speed is imparted to saidhollow toroidal one-piece polymeric bioreactor vessel, wherein theresonant frequency depends on the size and shape of the hollow toroidalone-piece polymeric bioreactor vessel, and wherein at least a portion ofan inner surface of said hollow toroidal one-piece polymeric bioreactorvessel is textured with a plurality of upstanding protuberances and saidhollow toroidal one-piece polymeric bioreactor vessel includes at leastone exit port for the removal of gaseous by-products.
 2. The apparatusin accordance with claim 1, wherein at least some of said upstandingprotuberances allow passage of sparging gas into said hollow toroidalone-piece polymeric bioreactor vessel.
 3. The apparatus in accordancewith claim 2, having the textured surface on a bottom of the hollowtoroidal one-piece polymeric bioreactor vessel.
 4. The apparatus inaccordance with claim 1, further comprising pre-molded baffles on atleast a portion of the inner surface of said hollow toroidal one-piecepolymeric bioreactor vessel.
 5. The apparatus in accordance with claim1, wherein the apparatus is fabricated from a substantially rigidplastic.
 6. The apparatus in accordance with claim 1, wherein theapparatus is fabricated from a substantially flexible plastic.
 7. Theapparatus in accordance with claim 1, further comprising means forilluminating the contents of said hollow toroidal one-piece polymericbioreactor vessel.
 8. The apparatus in accordance with claim 7 whereinsaid illumination means comprises at least one LED.
 9. The apparatus inaccordance with claim 1, further comprising means for simultaneouslyboth orbiting and rocking said hollow toroidal one-piece polymericbioreactor vessel.